Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Sibling species of the major malaria vector Anopheles gambiae display divergent preferences for aquatic breeding sites in southern Nigeria.

BACKGROUND: When integrated with insecticide-treated bed nets, larval control of Anopheles mosquitoes could fast-track reductions in the incidence of human malaria. However, larval control interventions may deliver suboptimal outcomes where the preferred breeding places of mosquito vectors are not well known. This study investigated the breeding habitat choices of Anopheles mosquitoes in southern Nigeria. The objective was to identify priority sites for mosquito larval management in selected urban and periurban locations where malaria remains a public health burden.  METHODS: Mosquito larvae were collected in urban and periurban water bodies during the wet-dry season interface in Edo, Delta, and Anambra States. Field-collected larvae were identified based on PCR gel-electrophoresis and amplicon sequencing, while the associations between Anopheles larvae and the properties and locations of water bodies were assessed using a range of statistical methods. RESULTS: Mosquito breeding sites were either man-made (72.09%) or natural (27.91%) and mostly drainages (48.84%) and puddles (25.58%). Anopheles larvae occurred in drainages, puddles, stream margins, and a concrete well, and were absent in drums, buckets, car tires, and a water-holding iron pan, all of which contained culicine larvae. Wild-caught Anopheles larvae comprised Anopheles coluzzii (80.51%), Anopheles gambiae sensu stricto (s.s.) (11.54%), and Anopheles arabiensis (7.95%); a species-specific PCR confirmed the absence of the invasive urban malaria vector Anopheles stephensi among field-collected larvae. Anopheles arabiensis, An. coluzzii, and An. gambiae s.s. displayed preferences for turbid, lowland, and partially sunlit water bodies, respectively. Furthermore, An. arabiensis preferred breeding sites located outside 500 m of households, whereas An. gambiae s.s. and An. coluzzii had increased detection odds in sites within 500 m of households. Anopheles gambiae s.s. and An. coluzzii were also more likely to be present in natural water bodies; meanwhile, 96.77% of An. arabiensis were in man-made water bodies. Intraspecific genetic variations were little in the dominant vector An. coluzzii, while breeding habitat choices of populations made no statistically significant contributions to these variations. CONCLUSION: Sibling malaria vectors in the An. gambiae complex display divergent preferences for aquatic breeding habitats in southern Nigeria. The findings are relevant for planning targeted larval control of An. coluzzii whose increasing evolutionary adaptations to urban ecologies are driving the proliferation of the mosquito, and An. arabiensis whose adults typically evade the effects of treated bed nets due to exophilic tendencies.

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes.

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life parameter data from genetically modified mosquitoes and comparing them to mosquitoes lacking transgenes from the same genetic background. This manuscript illustrates how to measure common life history traits in the mosquito Aedes aegypti, including fecundity, wing size and shape, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. The representative results quantify fitness costs associated with either a gene knock-out or a single insertion of a gene drive element. Standardizing how life parameter data are collected is important because such data may be used to compare the health of transgenic mosquitoes generated across studies or to model the transgene fixation rate in a simulated wild-type mosquito population. Although this protocol is specific for transgenic Aedes aegypti, the protocol may also be used for other mosquito species or other experimental treatment conditions, with the caveat that certain biological contexts may require special adaptations.

Assessing single-locus CRISPR/Cas9-based gene drive variants in the mosquito Aedes aegypti via single-generation crosses and modeling.

The yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika viruses. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus is amenable to both gene drive and appropriate expression of the antiviral effector. In our study, we used reverse engineering to target 2 intergenic genomic loci, which had previously shown to be highly permissive for antiviral effector gene expression, and we further investigated the use of 3 promoters (nanos, ß2-tubulin, or zpg) for Cas9 expression. We then quantified the accrual of insertions or deletions (indels) after single-generation crossings, measured maternal effects, and assessed fitness costs associated with various transgenic lines to model the rate of gene drive fixation. Overall, MGDrivE modeling suggested that when an autonomous gene drive is placed into an intergenic locus, the gene drive system will eventually be blocked by the accrual of gene drive blocking resistance alleles and ultimately be lost in the population. Moreover, while genomic locus and promoter selection were critically important for the initial establishment of the autonomous gene drive, it was the fitness of the gene drive line that most strongly influenced the persistence of the gene drive in the simulated population. As such, we propose that when autonomous CRISPR/Cas9-based gene drive systems are anchored in an intergenic locus, they temporarily result in a strong population replacement effect, but as gene drive-blocking indels accrue, the gene drive becomes exhausted due to the fixation of CRISPR resistance alleles.

Advancing towards the implementation of patient-centred care in Chile: An opportunity to effectively practice shared decision-making.

In Chile, local normative and guidelines place patient-centred care (PCC) as a desirable means and outcome for each level of health care. Thus, a definition of PCC is provided, and for the first time shared decision-making (SDM) is included as an intended practice. During the past five years the country has shown progress on the implementation of PCC. A large pilot study was conducted in one of the Metropolitan Health Services, and now the health authority is committed to escalate a PCC strategy nationwide. From the practice domain, most of the work is being placed on the training of health professionals. Patients' preparation for the clinical encounter is scarce, thereby limiting their potential to participate in their care. At the research domain, the country shows a strengthened agenda that has advanced from a diagnostic phase (including the exploration from social sciences) to a purposeful stage which involves the development of training programs, patient decision aids, international collaborations, and other PCC interventions. The country is now positioned to secure new initiatives to empower patients and allow them to take an active role, as a key component of PCC and SDM.

Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti.

The recent global Zika epidemics have revealed the significant threat that mosquito-borne viruses pose. There are currently no effective vaccines or prophylactics to prevent Zika virus (ZIKV) infection. Limiting exposure to infected mosquitoes is the best way to reduce disease incidence. Recent studies have focused on targeting mosquito reproduction and immune responses to reduce transmission. Previous work has evaluated the effect of insulin signaling on antiviral JAK/STAT and RNAi in vector mosquitoes. Specifically, insulin-fed mosquitoes resulted in reduced virus replication in an RNAi-independent, ERK-mediated JAK/STAT-dependent mechanism. In this work, we demonstrate that targeting insulin signaling through the repurposing of small molecule drugs results in the activation of both RNAi and JAK/STAT antiviral pathways. ZIKV-infected Aedes aegypti were fed blood containing demethylasterriquinone B1 (DMAQ-B1), a potent insulin mimetic, in combination with AKT inhibitor VIII. Activation of this coordinated response additively reduced ZIKV levels in Aedes aegypti. This effect included a quantitatively greater reduction in salivary gland ZIKV levels up to 11 d post-bloodmeal ingestion, relative to single pathway activation. Together, our study indicates the potential for field delivery of these small molecules to substantially reduce virus transmission from mosquito to human. As infections like Zika virus are becoming more burdensome and prevalent, understanding how to control this family of viruses in the insect vector is an important issue in public health.

Intrathoracic Inoculation of Zika Virus in Aedes aegypti.

Aedes aegypti mosquitoes are the main vectors of many medically relevant arthropod-borne (arbo) viruses, including Zika (ZIKV), dengue (DENV), and yellow fever (YFV). Vector competence studies with Ae. aegypti often involve challenging mosquitoes with an artificial bloodmeal containing virus and later quantifying viral titer or infectious plaque-forming units (PFU) in various mosquito tissues at relevant time points post-infection. However, Ae. aegypti mosquitoes are known to exhibit midgut infection and escape barriers (MIB and MEB, respectively), which influence the prevalence and titer of a disseminated infection and can introduce unwanted variability into studies analyzing tissues such as the salivary glands. To surmount this challenge, we describe herein a protocol for the intrathoracic inoculation of ZIKV in Ae. aegypti. This method bypasses the midgut, which leads to a more rapid and higher proportion of disseminated infections in comparison to oral challenge, and mosquitoes become infected with a consistent dose of virus. Our protocol is advantageous for studies that need a large sample size of infected mosquitoes, need to bypass the midgut, or are analyzing salivary gland infection or escape barriers. Graphic abstract: Cartoon depiction of Aedes aegypti intrathoracic inoculation. Figure made with Biorender.com.

Rediseño en el abordaje de personas con multimorbilidad crónica: desde la fragmentación al cuidado integral centrado en las personas / Redesign in the approach of people with multimorbidity chronic: from fragmentation to comprehensive care people-centered

En Chile, el 70% de la población de 15 años y más vive con multimorbilidad, es decir, con la presencia de dos o más condiciones crónicas de forma simultánea. El abordaje clásico de la cronicidad por programas en atención primaria de salud, con foco en la enfermedad, se expresa en cuidados fragmentados, ineficaces y muy alejados de los principios de centralidad en la persona, integralidad y continuidad del cuidado impulsados desde el modelo de atención integral de salud familiar y comunitario (MAIS). La estrategia de cuidado integral centrado en las personas para la promoción, prevención y manejo de la cronicidad en contexto de multimorbilidad (ECICEP), se constituye en una respuesta a esta problemática.La multimorbilidad representa un desafío de gran envergadura en el rediseño desde una atención fragmentada hacia el cuidado integral centrado en la persona. Implica un proceso de gestión del cambio, en donde es necesario sensibilizar en la urgencia y sentido del cambio, estratificar a la población según riesgo, capacitar a los equipos de salud, reorganizar los procesos administrativos (agendamiento, registro clínico) y clínicos (ingreso y control integral, planes de cuidado consensuados, gestión del cuidado, seguimiento a distancia, automanejo), así como favorecer el liderazgo y acompañamiento del cambio y el trabajo colaborativo en red.Este proceso requiere voluntad política, con sentido de urgencia del cambio y gradualidad, para que su instalación sea eficiente y respetuosa. Por ello, se inicia el proceso con las personas de alta complejidad, que son quienes tienen más riesgo de hospitalizaciones evitables y otras complicaciones

In Chile, 70% of the population aged 15 years and over lives with multimorbidity, that is, with the presence of two or more chronic conditions simultaneously. The classic approach to chronicity by programs in primary health care, with a focus on the disease, is expressed in fragmented care, ineffective and far removed from the principles of person-centeredness, comprehensiveness and continuity of care promoted by the Comprehensive Family and Community Health Care Model (MAIS). The People-Centered Integrated Care Strategy for the Promotion, Prevention and Management of Chronicity in the Context of Multimorbidity (ECICEP) is a response to this problem. Chronic multimorbidity represents a major challenge in the redesign from fragmented care to comprehensive person-centered care. It implies a process of change management, in which it is necessary to raise awareness of the urgency and sense of change, stratify the population according to risk, train health teams, reorganize administrative (scheduling, clinical records) and clinical processes (admission and comprehensive control, consensual care plans, care management, remote follow-up, self-management), as well as promoting leadership and accompaniment of change, networking and intersectoral coordination. This process requires political will, with a sense of urgency of change and gradualness, so that its installation is efficient and respectful. For this reason, the process begins with highly complex patients, who are at the greatest risk of avoidable hospitalizations and other complications.

Nootkatone Is an Effective Repellent against Aedes aegypti and Aedes albopictus.

We tested a nootkatone product for insecticide activity against the most prominent vectors of Zika virus (ZIKV), Aedes aegypti, and Aedes albopictus. We tested the permethrin-resistant (PERM-R) Vergel strain of A. aegypti and the permethrin-susceptible (PERM-S) New Orleans strain of A. aegypti to determine if insecticide resistance affected their susceptibility to nootkatone. Bottle bioassays showed that the PERM-S strain (New Orleans) was more susceptible to nootkatone than the confirmed A. aegypti permethrin-resistant (PERM-R) strain, Vergel. The A. albopictus strain ATM-NJ95 was a known PERM-S strain and Coatzacoalcos permethrin susceptibility was unknown but proved to be similar to the ATM-NJ95 PERM-S phenotype. The A. albopictus strains (ATM-NJ95 and Coatzacoalcos) were as susceptible to nootkatone as the New Orleans strain. Bottle bioassays conducted with ZIKV-infected mosquitoes showed that the New Orleans (PERM-S) strain was as susceptible to nootkatone as the mock-infected controls, but the PERM-R strain was less susceptible to nootkatone than the mock-infected controls. Repellency/irritancy and biting inhibition bioassays (RIBB) of A. aegypti determined whether the nootkatone-treated arms of three human subjects prevented uninfected A. aegypti mosquitoes from being attracted to the test subjects and blood-feeding on them. The RIBB analyses data calculated the spatial activity index (SAI) and biting inhibition factor (BI) of A. aegypti at different nootkatone concentrations and then compared the SAI and BI of existing repellency products. We concluded that nootkatone repelled mosquitoes at a rate comparable to 7% DEET or 5% picaridin and has the potential to be an efficacious repellent against adult A. aegypti mosquitoes.

The Genetic Basis for Salivary Gland Barriers to Arboviral Transmission.

Arthropod-borne viruses (arboviruses) infect mosquito salivary glands and then escape to saliva prior to virus transmission. Arbovirus transmission from mosquitoes can be modulated by salivary gland infection barriers (SGIBs) and salivary gland escape barriers (SGEBs). We determined the influence of SGIBs and SGEBs by estimating the quantitative genetic contributions of Aedes aegypti half-sib families (Mapastepec, Mexico) infected with three dengue 2 (DENV2), two chikungunya (CHIKV), and two Zika (ZIKV) genotypes. We determined virus titer per salivary gland and saliva at seven days post-infection and virus prevalence in the half-sib population. CHIKV or ZIKV genotypes did not present SGIB, whereas DENV2 genotypes showed low rates of SGIB. However, virus titer and prevalence due to additive genetic factors in the half-sib family displayed a significant narrow-sense heritability (h2) for SGIB in two of the three DENV2 genotypes and one CHIKV and one ZIKV genotype. SGEBs were detected in all seven virus strains: 60-88% of DENV2 and 48-62% of CHIKV or ZIKV genotype infections. SGEB h2 was significant for all CHIKV or ZIKV genotypes but not for any of the DENV2 genotypes. SGIBs and SGEBs exhibited classical gene-by-gene interaction dynamics and are influenced by genetic factors in the mosquito and the virus.

The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic Aedes aegypti.

The resurgence of arbovirus outbreaks across the globe, including the recent Zika virus (ZIKV) epidemic in 2015-2016, emphasizes the need for innovative vector control methods. In this study, we investigated ZIKV susceptibility to transgenic Aedes aegypti engineered to target the virus by means of the antiviral small-interfering RNA (siRNA) pathway. The robustness of antiviral effector expression in transgenic mosquitoes is strongly influenced by the genomic insertion locus and transgene copy number; we therefore used CRISPR/Cas9 to re-target a previously characterized locus (Chr2:321382225) and engineered mosquitoes expressing an inverted repeat (IR) dsRNA against the NS3/4A region of the ZIKV genome. Small RNA analysis revealed that the IR effector triggered the mosquito's siRNA antiviral pathway in bloodfed females. Nearly complete (90%) inhibition of ZIKV replication was found in vivo in both midguts and carcasses at 7 or 14 days post-infection (dpi). Furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that Ae. aegypti innate immunity can be co-opted to engineer mosquitoes resistant to ZIKV.

Control of RNA viruses in mosquito cells through the acquisition of vDNA and endogenous viral elements.

Aedes aegypti transmit pathogenic arboviruses while the mosquito itself tolerates the infection. We examine a piRNA-based immunity that relies on the acquisition of viral derived cDNA (vDNA) and how this pathway discriminates between self and non-self. The piRNAs derived from these vDNAs are essential for virus control and Piwi4 has a central role in the pathway. Piwi4 binds preferentially to virus-derived piRNAs but not to transposon-targeting piRNAs. Analysis of episomal vDNA from infected cells reveals that vDNA molecules are acquired through a discriminatory process of reverse-transcription and recombination directed by endogenous retrotransposons. Using a high-resolution Ae. aegypti genomic sequence, we found that vDNAs integrated in the host genome as endogenous viral elements (EVEs), produce antisense piRNAs that are preferentially loaded onto Piwi4. Importantly, EVE-derived piRNAs are specifically loaded onto Piwi4 to inhibit virus replication. Thus, Ae. aegypti employs a sophisticated antiviral mechanism that promotes viral persistence and generates long-lasting adaptive immunity.

Estimation of DENV-2 Transmission as a Function of Site-Specific Entomological Parameters from Three Cities in Colombia.

BACKGROUND: Measuring dengue virus transmission in endemic areas is a difficult task as many variables drive transmission, and often are not independent of one another. OBJECTIVES: We aimed to determine the utility of vectorial capacity to explain the observed dengue infection rates in three hyperendemic cities in Colombia, and tested hypotheses related to three variables: mosquito density, effective vector competence, and biting rate. METHODS: We estimated two of the most influential entomological variables related to cumulative vectorial capacity, which is a modification of the traditional vectorial capacity equation, of three Colombian mosquito populations. Laboratory studies were undertaken to measure vector competence and man biting rate of local mosquito populations. In addition, the assessment of cumulative vectorial capacity also incorporated site-specific estimations of mosquito density and the probability of daily survival from previous studies conducted in those cities. FINDINGS: We found that the biting rates and mosquito infection rates differed among populations of mosquitoes from these three cities, resulting in differences in the site-specific measures of transmission potential. Specifically, we found that using site-specific entomological measures to populate the cumulative vectorial capacity equation was best at recapitulating observed mosquito infection rates when mosquito density was discounted compared to when we incorporated site-specific density measures. CONCLUSIONS: Specific mosquito-biting rate is likely sufficient to explain transmission differences in these three cities, confirming that this parameter is a critical parameter when predicting and assessing dengue transmission in three Colombian cities with different field observed transmission patterns.

Analysis of Salivary Glands and Saliva from Aedes albopictus and Aedes aegypti Infected with Chikungunya Viruses.

Chikungunya virus (CHIKV) is a medically important mosquito-borne virus transmitted to humans by infected Aedes (Stegomyia) species. In 2013⁻2014, Ae. aegypti transmitted CHIKV to humans in the Caribbean and in 2005⁻2006, Ae. albopictus transmitted CHIKV on La Réunion Island (Indian Ocean basin). CHIKV LR2006 OPY1 from the La Réunion epidemic was associated with a mutation (E1:A226V) in the viral E1 glycoprotein that enhanced CHIKV transmission by Ae. albopictus. CHIKV R99659 from the Caribbean outbreak did not have the E1:A226V mutation. Here, we analyzed the salivary glands and saliva of Ae. albopictus strains from New Jersey, Florida, Louisiana and La Réunion after infection with each virus to determine their transmission potential. We infected the Ae. albopictus strains with blood meals containing 3⁻7 × 107 PFU/mL of each virus and analyzed the mosquitoes nine days later to maximize infection of their salivary glands. All four Ae. albopictus strains were highly susceptible to LR2006 OPY1 and R99659 viruses and their CHIKV disseminated infection rates (DIR) were statistically similar (p = 0.3916). The transmission efficiency rate (TER) was significantly lower for R99659 virus compared to LR2006 OPY1 virus in all Ae. albopictus strains and Ae. aegypti (Poza Rica) (p = 0.012) suggesting a salivary gland exit barrier to R99659 virus not seen with LR2006 OPY1 infections. If introduced, LR2006 OPY1 virus poses an increased risk of transmission by both Aedes species in the western hemisphere.

RNA Structure Duplication in the Dengue Virus 3' UTR: Redundancy or Host Specificity?

Flaviviruses include a diverse group of medically important viruses that cycle between mosquitoes and humans. During this natural process of switching hosts, each species imposes different selective forces on the viral population. Using dengue virus (DENV) as model, we found that paralogous RNA structures originating from duplications in the viral 3' untranslated region (UTR) are under different selective pressures in the two hosts. These RNA structures, known as dumbbells (DB1 and DB2), were originally proposed to be enhancers of viral replication. Analysis of viruses obtained from infected mosquitoes showed selection of mutations that mapped in DB2. Recombinant viruses carrying the identified variations confirmed that these mutations greatly increase viral replication in mosquito cells, with low or no impact in human cells. Use of viruses lacking each of the DB structures revealed opposite viral phenotypes. While deletion of DB1 reduced viral replication about 10-fold, viruses lacking DB2 displayed a great increase of fitness in mosquitoes, confirming a functional diversification of these similar RNA elements. Mechanistic analysis indicated that DB1 and DB2 differentially modulate viral genome cyclization and RNA replication. We found that a pseudoknot formed within DB2 competes with long-range RNA-RNA interactions that are necessary for minus-strand RNA synthesis. Our results support a model in which a functional diversification of duplicated RNA elements in the viral 3' UTR is driven by host-specific requirements. This study provides new ideas for understanding molecular aspects of the evolution of RNA viruses that naturally jump between different species.IMPORTANCE Flaviviruses constitute the most relevant group of arthropod-transmitted viruses, including important human pathogens such as the dengue, Zika, yellow fever, and West Nile viruses. The natural alternation of these viruses between vertebrate and invertebrate hosts shapes the viral genome population, which leads to selection of different viral variants with potential implications for epidemiological fitness and pathogenesis. However, the selective forces and mechanisms acting on the viral RNA during host adaptation are still largely unknown. Here, we found that two almost identical tandem RNA structures present at the viral 3' untranslated region are under different selective pressures in the two hosts. Mechanistic studies indicated that the two RNA elements, known as dumbbells, contain sequences that overlap essential RNA cyclization elements involved in viral RNA synthesis. The data support a model in which the duplicated RNA structures differentially evolved to accommodate distinct functions for viral replication in the two hosts.

Efectividad de un modelo de manejo de pacientes con hipertensión y diabetes tipo II en atención primaria / Effectiveness of a case management intervention for high blood pressure and type II diabetes in primary health care

Background: Adequate management of high blood pressure (HBP) and Type 2 Diabetes (DM2) is a challenge to the healthcare system in Chile. Aim: To evaluate the effectiveness of a case management (CM) approach to manage HBP and DMII at Primary Healthcare (PHC) level, headed by healthcare technicians with the supervision of registered nurses. Material and Methods: Two primary health care centers were selected. In one the case management approach was used and the other continued with the usual care model. Patients with HBP or DM2 were selected to participate in both centers. The main outcomes were changes blood pressure and glycosylated hemoglobin levels. Results: Three hundred twenty-eight patients were allocated to the intervention group and 316 to control group. At the baseline evaluation, participants at the control health center had better systolic and diastolic BP levels (SBP and DBP), but no difference in glycosylated hemoglobin. After twelve months the adjusted mean difference in HBP patients for SBP was −0.93 (95% conficence intervals (CI) −5.49,3.63) and for DBP was 1.78 (95%CI −2.89,6.43). Among HBP+DMII patients, the mean difference for SBP was −0.51 (95% −0.52,0.49) and for DBP was −3.39 (95%CI −6.07, −0.7). No differences in glycosylated hemoglobin were observed. In a secondary analysis, the intervention group showed a statistically significant higher SBP and DBP reduction than the control group. Conclusions: The case management approach tested in this study had promissory results among patients with high blood pressure.

Demonstration of efficient vertical and venereal transmission of dengue virus type-2 in a genetically diverse laboratory strain of Aedes aegypti.

Aedes aegypti is the primary mosquito vector of dengue viruses (DENV; serotypes 1-4). Human-mosquito transmission cycles maintain DENV during epidemics but questions remain regarding how these viruses survive when human infections and vector abundance are minimal. Aedes mosquitoes can transmit DENV within the vector population through two alternate routes: vertical and venereal transmission (VT and VNT, respectively). We tested the efficiency of VT and VNT in a genetically diverse laboratory (GDLS) strain of Ae. aegypti orally infected with DENV2 (Jamaica 1409). We examined F1 larvae from infected females generated during the first and second gonotrophic cycles (E1 and E2) for viral envelope (E) antigen by amplifying virus in C6/36 cells and then performing an indirect immunofluorescence assay (IFA). RT-PCR/nested PCR analyses confirmed DENV2 RNA in samples positive by IFA. We observed VT of virus to larvae and adult male progeny and VNT of virus to uninfected virgin females after mating with males that had acquired virus by the VT route. We detected no DENV2 in 30 pools (20 larvae/pool) of F1 larvae following the first gonotrophic cycle, suggesting limited virus dissemination at 7 days post-infection. DENV2 was detected by IFA in 27 of 49 (55%) and 35 of 51 (68.6%) F1 larval pools (20 larvae/pool) from infected E2 females that received a second blood meal without virus at 10 or 21 days post-infection (E2-10d-F1 and E2-21-F1), respectively. The minimum filial infection rates by IFA for E2-10d-F1 and E2-21d-F1 mosquitoes were 1:36 and 1:29, respectively. The VNT rate from E2-10d-F1 males to virgin (uninfected) GDLS females was 31.6% (118 of 374) at 8 days post mating. Twenty one percent of VNT-infected females receiving a blood meal prior to mating had disseminated virus in their heads, suggesting a potential pathway for virus to re-enter the human-mosquito transmission cycle. This is the first report of VNT of DENV by male Ae. aegypti and the first demonstration of sexual transmission in Aedes by naturally infected males. Our results demonstrate the potential for VT and VNT of DENV in nature as mechanisms for virus maintenance during inter-epidemic periods.

Modelo de atención integral en salud familiar y comunitaria en la atención primaria chilena

[RESUMEN]. Objetivo. Describir el estado actual de la implementación de Modelo de Atención Integral en Salud Familiar y Comunitaria (MAIS) en la atención primaria de Chile. Métodos. Estudio transversal que evaluó la implementación del MAIS en un total de 1 263 establecimientos de atención primaria. Por medio de correlaciones se estudió la relación entre la autoevaluación (interna) y la evaluación de los servicios de salud (externa) para cada centro. Con los análisis multinivel se evaluaron los factores de establecimientos, comunas y regiones asociados con el nivel de implementación del MAIS. Resultados. La correlación entre autoevaluación interna y la evaluación externa de la implementación total del MAIS fue muy alta (0,819, p < 0,001). El eje tecnología presentó mayor implementación (83,0% de cumplimiento) y enfoque familiar (37,8% de cumplimiento), el menor. Los centros de salud familiar, las comunas urbanas, aquellas con mayor número de inscritos y con menor índice de pobreza, fueron los establecimientos que presentaron mayor implementación. No se identificó una asociación estadísticamente significativa entre la implementación del MAIS y los gastos comunales totales (p = 0,132) ni específicos de salud (p = 0,244). Conclusiones. La mayoría de los establecimientos de salud de atención primaria han evaluado el nivel de implementación del. MAIS. Las estrategias de acompañamiento para su implementación son prioritarias para establecimientos de atención primaria ubicados en zonas rurales y con bajo número de usuarios inscritos. Aún persiste el desafío de avanzar en la instalación del enfoque familiar y la calidad del cuidado como centro de la atención de salud.

[ABSTRACT]. Objective. Describe the current status of the implementation of the Model of Comprehensive Care in Family and Community Health (MAIS, by its acronym in Spanish) in primary care in Chile. Methods. Cross-sectional study that evaluated the implementation of MAIS in a total of 1 263 primary care facilities. Through correlations, the relationship between internal self-evaluation and external evaluation of health services for each center was studied. The factors of facilities, communes and regions associated with the level of implementation of the MAIS were evaluated with multilevel analyses. Results. The correlation between internal self-evaluation and the external evaluation of the total implementation of the MAIS was very high (0.819, p <0.001). The technology axis presented the highest implementation (83.0% compliance), and family focus the lowest (37.8% compliance). The facilities with the highest implementation were family health centers, the urban communes, those with the highest number of enrollees and those with the lowest poverty index. A statistically significant association was not identified between the implementation of the MAIS and the total community expenses (p = 0.122) nor specific health expenditures (p = 0.244). Conclusions. Most of the primary care health facilities have evaluated the level of implementation of the MAIS. The accompanying strategies for its implementation are priorities for primary care facilities located in rural areas and with a low number of registered users. Improving the family focus and the quality of care —key aspects of health care— are still a challenge.

[RESUMO]. Objetivo. Descrever o estado atual da implementação do Modelo de Assistência Integral em Saúde da Família e da Comunidade (MAIS) na atenção primária no Chile. Métodos. Estudo transversal que avaliou a implementação do MAIS em um total de 1 263 estabelecimentos de atenção primária. Por meio de correlações, foi estudada a relação entre a autoavaliação (interna) e a avaliação dos serviços de saúde (externa) para cada centro. Os fatores dos estabelecimentos, municípios e regiões associados ao nível de implementação do MAIS foram avaliados com análises multiníveis. Resultados. A correlação entre a autoavaliação interna e a avaliação externa da implementação total do MAIS foi muito alta (0,819, p <0,001). O eixo tecnológico apresentou maior implementação (83,0% de cumprimento) e foco familiar o menor (37,8% de cumprimento). Os centros de saúde da família, as comunas urbanas, aqueles com maior número de inscritos e com o menor índice de pobreza, foram os estabelecimentos que apresentaram a maior implementação. Não foi identificada associação estatisticamente significativa entre a implementação do MAIS e as despesas totais da comunidade (p = 0,122) nem gastos específicos com saúde (p = 0,244). Conclusões. A maioria dos estabelecimentos de atenção primária avaliaram o nível de implementação do MAIS. As estratégias de acompanhamento para sua implementação são prioritárias para estabelecimentos de atenção primária em áreas rurais e com baixo número de usuários cadastrados. Enfatiza-se o desafio de avançar na instalação do enfoque familiar e na qualidade de atenção.

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes.

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.

[Effectiveness of a case management intervention for high blood pressure and type II diabetes in primary health care]. / Efectividad de un modelo de manejo de pacientes con hipertensión y diabetes tipo II en atención primaria.

BACKGROUND: Adequate management of high blood pressure (HBP) and Type 2 Diabetes (DM2) is a challenge to the healthcare system in Chile. AIM: To evaluate the effectiveness of a case management (CM) approach to manage HBP and DMII at Primary Healthcare (PHC) level, headed by healthcare technicians with the supervision of registered nurses. MATERIAL AND METHODS: Two primary health care centers were selected. In one the case management approach was used and the other continued with the usual care model. Patients with HBP or DM2 were selected to participate in both centers. The main outcomes were changes blood pressure and glycosylated hemoglobin levels. RESULTS: Three hundred twenty-eight patients were allocated to the intervention group and 316 to control group. At the baseline evaluation, participants at the control health center had better systolic and diastolic BP levels (SBP and DBP), but no difference in glycosylated hemoglobin. After twelve months the adjusted mean difference in HBP patients for SBP was -0.93 (95% conficence intervals (CI) -5.49,3.63) and for DBP was 1.78 (95%CI -2.89,6.43). Among HBP+DMII patients, the mean difference for SBP was -0.51 (95% -0.52,0.49) and for DBP was -3.39 (95%CI -6.07, -0.7). No differences in glycosylated hemoglobin were observed. In a secondary analysis, the intervention group showed a statistically significant higher SBP and DBP reduction than the control group. CONCLUSIONS: The case management approach tested in this study had promissory results among patients with high blood pressure.